A fundamental component of energy metabolism is glucose transport. The transport of glucose across cell membranes is essential to metabolic processes, including the maintenance of a relatively constant blood glucose concentration and the delivery of glucose to peripheral tissues for storage and utilization. As cell membranes are essentially impermeable to glucose, the movement of glucose across membranes must be accomplished by protein transporters (Brown, J. Inherit. Metab. Dis., 2000, 23, 237-246).
Mediated glucose transport occurs in two forms, secondary active transport and facilitated transport. In cells where glucose is rapidly metabolized, the concentration gradient across the plasma membrane is used to drive facilitated transport, and an active mechanism is not required. Secondary active transport of glucose enables cells to transport glucose against a concentration gradient. This mechanism involves cotransport of glucose and sodium ions across the apical surface of the cells and the energy is provided by the sodium gradient maintained by the sodium/potassium ATPase in the basolateral membrane. Efflux of glucose from the cells into the circulation is then mediated by a facilitative transporter (Brown, J. Inherit. Metab. Dis., 2000, 23, 237-246; Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18).
Secondary active transport of glucose operates in the mucosal cells of the intestine and the proximal tubular cells of the kidney and functions to ensure efficient uptake of dietary glucose and minimal urinary loss. Plasma glucose is normally filtered in the kidney in the glomerulus and actively reabsorbed in the proximal tubule. Glucose is essentially completely reabsorbed from the urine in the proximal tubule of the kidney through the action of the sodium-glucose cotransporters (SGLTs) located in the brush border membrane (BBM). Comparison of the glucose transport properties of proximal tubule BBM vesicles prepared from the outer cortex and the outer medulla of rabbit kidney revealed the presence of two distinct sodium-coupled D-glucose transport systems. The outer cortex preparation exhibited a low-affinity/high-capacity activity (Km=6 mM), whereas the outer medulla displayed a high-affinity/low-capacity activity (Km=0.35 mM) (Turner and Moran, Am. J. Physiol. Endocrinol. Metab., 1982, 242, F406-414; Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18). Further characterization of the renal outer cortical BBM transport system revealed a glucose to sodium coupling ratio of 1:1, whereas the ratio is 2:1 in vesicles isolated from the outer medullary tissue (Turner and Moran, J. Membr. Biol., 1982, 67, 73-80).
Isolation of nucleic acid molecules encoding SGLTs confirmed the presence of multiple transport systems. A cDNA encoding human SGLT2 (also known as solute carrier family 2, member 5, Na-dependent glucose cotransporter 2 or SLC2A5) was identified in a screen for sodium cotransporter-like sequences in a cDNA library prepared from human kidney (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; Wells et al., Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465). Human SGLT2 localizes to chromosome 16p11.2 (Wells et al., Genomics, 1993, 17, 787-789). Subsequent investigations of human SGLT2 revealed that has functional properties characteristic of a low-affinity, sodium-dependent glucose cotransporter.
Studies of human SGLT2 injected into Xenopus oocytes demonstrated that this protein mediates sodium-dependent transport of D-glucose and α-methyl-D-glucopyranoside (α-MeGlc; a glucose analog) with a Km value of 1.6 mM for α-MeGlc and a sodium to glucose coupling ratio of 1:1 (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; You et al., J. Biol. Chem., 1995, 270, 29365-29371). This transport activity was suppressed by phlorizin, a plant glycoside that binds to the glucose site but is not transported and thus inhibits SGLTs (You et al., J. Biol. Chem., 1995, 270, 29365-29371). These findings indicated that SGLT2 is responsible for the low-affinity transport observed in BBM vesicle preparations from rabbit kidney outer cortex.
The tissue distribution of SGLT2 further suggested that this cotransporter is the kidney low-affinity glucose transporter. Northern blotting revealed that human SGLT2 is primarily expressed in kidney, and in situ hybridization of a human SGLT2 probe to rat kidney tissue demonstrated that SGLT2 is expressed in the proximal tubule S1 segments in the outer cortex (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; Wells et al., Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465). This localization pattern distinguishes SGLT2 from SGLT1, the high-affinity/low-capacity sodium/glucose transporter that is expressed in the proximal tubule S3 segments of the outer medulla, where it is appropriately positioned to reabsorb the remainder of filtered glucose not reabsorbed by SGLT2 in the proximal tubule S1 segments.
Rat SGLT2, like human SGLT2, is strongly expressed in proximal S1 segments and this expression is developmentally regulated, with expression appearing on embryonic day 17, gradually increasing until day 19 and subsequently decreasing between day 19 and birth. Interestingly, rat SGLT2 mRNA is 2.6 kb before birth and 2.2 kb after birth, suggesting the presence of a different splice variant in embryonic kidney compared to the adult (You et al., J. Biol. Chem., 1995, 270, 29365-29371).
The transport properties of rat SGLT2, i.e Km of 3.0 mM and sodium to glucose coupling of 1:1, are also characteristic of a kidney cortical low-affinity transport system. Hybrid depletion studies in which rat kidney superficial cortex mRNA was mixed with an antisense oligonucleotide corresponding to the 5′ portion of the rat SGLT2 coding region completely suppressed the uptake of α-MeGlc in Xenopus oocytes into which the mRNA/oligonucleotide mix was injected. An antisense oligonucleotide targeted to SGLT1 had no effect on the uptake of α-MeGlc. These data demonstrate that the α-MeGlc uptake was entirely due to the expression of rat SGLT2 and support the proposal that SGLT2 is the major kidney cortical low affinity glucose transporter (You et al., J. Biol. Chem., 1995, 270, 29365-29371).
A second low-affinity SGLT, named SAAT-pSGLT2, was isolated from porcine kidney cells and was initially proposed to be the main low-affinity glucose transporter. However, further studies have revealed that the molecular characteristics of SAAT-pSGLT2 differ from those of SGLT2 and consequently SAAT-pSGLT2 has been renamed SGLT3 (Kong et al., J. Biol. Chem., 1993, 268, 1509-1512; Mackenzie et al., J. Biol. Chem., 1996, 271, 32678-32683; Mackenzie et al., J. Biol. Chem., 1994, 269, 22488-22491; You et al., J. Biol. Chem., 1995, 270, 29365-29371). Whether SGLT3 contributes to glucose reabsorption in a physiologically relevant manner is unclear.
The importance of SGLT2 function was demonstrated in hepatocyte nuclear factor 1α (HNF 1α)-deficient animals, which are diabetic and also suffer from a renal Fanconi syndrome characterized by urinary glucose loss. HNF 1α is a transcriptional activator expressed in liver, kidney, pancreas and intestine. The renal defect in these mice is due to an 80-90% reduction in SGLT2 expression. Thus, HNF1α is one gene product that controls SGLT2 expression, which is essential to proper glucose reabsorption in vivo (Pontoglio et al., EMBO Rep., 2000, 1, 359-365).
Reduction of SGLT2 mRNA was also observed upon exposure of mouse kidney cortical cells to cadmium, along with inhibition of sodium-dependent uptake of the glucose analog α-MeGlc. Interestingly, while both SGLT1 and SGLT2 mRNA were decreased in mouse kidney cortical cells exposed to cadmium, SGLT3 mRNA was upregulated, suggesting that individual SGLT species are not regulated in a similar manner (Tabatabai et al., Toxicol. Appl. Pharmacol., 2001, 177, 163-173). Changes in glucose or sodium filtrated rate also modulate the expression of sodium-glucose transporter mRNA. Diabetic rats with glycosuria and rats fed a high sodium diet exhibited increased SGLT2 expression in the renal proximal tubule. The finding that SGLT1 levels in these rats were not altered to the same extent as SGLT2 levels further supports the hypothesis that the cotransporters are differentially regulated (Vestri et al., J. Membr. Biol., 2001, 182, 105-112).
Although studies of SGLT function and localization in multiple mammalian species, including rat, mouse, pig, rabbit and dog, indicated that SGLT2 is the low-affinity renal SGLT, the identity of the human SGLT responsible for glucose reabsorption across the brush border of the human proximal tubule remained unclear. The lack of information describing SGLT protein localization in renal brush border further hindered the identification of the human low-affinity SGLT. Molecular genetic analysis of SGLT1 and SGLT2 indicated that a genetic alteration in the SGLT2 gene is a likely cause of renal glycosuria, a condition characterized by elevated excretion of glucose in the urine (Hediger et al., Klin. Wochenschr., 1989, 67, 843-846). Direct evidence of SGLT function in the reabsorption of glucose came from analysis of the SGLT2 gene in a patient with congenital isolated renal glucosuria. Sequence analysis revealed a homozygous nonsense mutation in exon 11 of the SGLT2 gene leading to the formation of a truncated protein which is predicted to lack cotransport function (van den Heuvel et al., Hum. Genet., 2002, 111, 544-547).
Whereas SGLT2 deficiency leads to inhibited reabsorption of glucose, SGLT2 elevation potentially allows for increased glucose uptake and is observed in metastatic lesions of lung cancer. Quantitation of SGLT2 gene expression revealed no significant difference between normal lung tissue and primary lung cancer. However, the metatstatic lesions of both the liver and lymph node exhibited significantly higher expression of SGLT2 (Ishikawa et al., Jpn. J. Cancer Res., 2001, 92, 874-879). This finding is significant in light of evidence that different clinical tumors show significantly increased glucose uptake in vivo compared to normal tissue. Such a change in metabolism confers an advantage to tumor cells which allows them to survive and invade. Furthermore, glucose uptake correlates with tumor aggressiveness and prognosis (Dang and Semenza, Trends Biochem. Sci., 1999, 24, 68-72).
Diabetes is a disorder characterized by hyperglycemia due to deficient insulin action. Chronic hyperglycemia is a major risk factor for diabetes-associated complications, including heart disease, retinopathy, nephropathy and neuropathy. As the kidneys play a major role in the regulation of plasma glucose levels, renal glucose transporters are becoming attractive drug targets (Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18). Synthetic agents that are derived from phlorizin, a specific inhibitor of sodium/glucose transporters, have been designed and include T-1095, and its metabolically active form T-1095A (Tsujihara et al., J. Med. Chem., 1999, 42, 5311-5324). Phlorizin, T-1095 and T-1095A all inhibited sodium-dependent glucose uptake in brush border membranes prepared from normal and diabetic rat kidney, rat small intestine, mouse kidney and dog kidney, as well as in Xenopus oocytes injected with human SGLT mRNA (Oku et al., Diabetes, 1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192). These agents have been tested as antidiabetic compounds in laboratory animals with genetic and streptozotocin-induced diabetes. In these models, administration of these compounds inhibited renal SGLT activity, increased urinary glucose excretion and improved glucose tolerance, hyperglycemia and hypoinsulemia (Arakawa et al., Br. J. Pharmacol., 2001, 132, 578-586; Oku et al., Diabetes, 1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192). Prolonged treatment of db/db mice with T-1095 yielded similar results and also almost completely suppressed the increase of urinary albumin and improved renal glomeruli pathology, indicating a beneficial influence on renal disfunction and a protective effect against nephropathy, respectively (Arakawa et al., Br. J. Pharmacol., 2001, 132, 578-586). Diabetic nephropathy is the most common cause of end-stage renal disease that develops in many patients with diabetes. In Zucker diabetic fatty rats, long-term treatment with T-1095 lowered both fed and fasting glucose levels to near normal ranges. Also observed were recovered hepatic glucose production and glucose utilization rates without a significant improvement in skeletal muscle glucose utilization rate, indicating that hyperglycemia contributes to insulin resistance in hepatic and adipose tissue in this rat model of diabetes. These results further suggest that glucotoxicity, which results from long-term hyperglycemia, induces tissue-dependent insulin resistance in diabetic patients (Nawano et al., Am. J. Physiol. Endocrinol. Metab., 2000, 278, E535-543).
Other SGLT2 inhibiting compounds are known in the art, such as the c-aryl glucosides disclosed in U.S. Pat. No. 6,414,126, which are inhibitors of sodium dependent glucose transporters found in the intestine and kidney and are proposed to treat diabetes, hyperglycemia and related diseases when used alone or in combination with other antidiabetic agents (Ellsworth et al., 2002).
The US pre-grant publication 20030055019 discloses isolated mutant proteins selected from a group which includes SGLT2, the corresponding nucleic acid molecules encoding said mutant proteins, isolated antisense derivatives of the nucleic acid sequences encoding said mutant proteins, as well as methods of delivering said antisense nucleic acid derivatives to treat or prevent hypertension, diabetes, insulin sensitivity, obesity, dyslipidemia and stroke. This application also discloses the antisense molecules may be DNA or RNA or a chimeric mixture, single-stranded or double-stranded or may comprise a ribozyme or catalytic RNA (Shimkets, 2003).
The European Patent Applications EP 1 293 569 and EP 1 308 459 disclose a polynucleotide comprising a protein-coding region of the nucleotide sequence of any one of a group of sequences which includes a nucleic acid sequence encoding human SGLT2, an oligonucleotide comprising at least 15 nucleotides complementary to the nucleotide sequence or to a complementary strand thereof and an antisense polynucleotide against the disclosed polynucleotide or a part thereof. These applications disclose the use of said antisense polynucleotides for suppressing the expression of a polypeptide of the invention and for gene therapy (Isogai et al., 2003; Isogai et al., 2003).
Although phlorizin and its derivatives are potent inhibitors of sodium-glucose cotransporters, these agents do not specifically inhibit a single species of SGLT, thus all SGLTs in all tissues are affected. Thus, there remains a need for therapeutic compounds that targets specific SGLT species. Antisense technology is an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of SGLT2 expression.
The present invention provides compounds and methods for modulating SGLT2 expression.